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Abstract Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour‐specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low‐cost methods remains challenging, especially in early‐stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian‐cancer‐specific aberrations of DNA methylation from routinely‐collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high‐resolution melt to analyze locus‐specific DNA methylation patterns on a molecule‐by‐molecule basis at or near single CpG‐site resolution at a fraction (

Abstract Background ARID1A, a tumor suppressor gene encoding BAF250, a protein participating in chromatin remodeling, is frequently mutated in endometrium-related malignancies, including ovarian or uterine clear cell carcinoma (CCC) and endometrioid carcinoma (EMCA). However, how ARID1A mutations alter downstream signaling to promote tumor development is yet to be established. Methods We used RNA-sequencing (RNA-seq) to explore transcriptomic changes in isogenic human endometrial epithelial cells after deleting ARID1A. Chromatin immunoprecipitation sequencing (ChIP-seq) was employed to assess the active or repressive histone marks on DUSP4 promoter and regulatory regions. We validated our findings using genetically engineered murine endometroid carcinoma models, human endometroid carcinoma tissues, and in silico approaches. Results RNA-seq revealed the downregulation of the MAPK phosphatase dual-specificity phosphatase 4 (DUSP4) in ARID1A-deficient cells. ChIP-seq demonstrated decreased histone acetylation marks (H3K27Ac, H3K9Ac) on DUSP4 regulatory regions as one of the causes for DUSP4 downregulation in ARID1A-deficient cells. Ectopic DUSP4 expression decreased cell proliferation, and pharmacologically inhibiting the MAPK pathway significantly mitigated tumor formation in vivo. Conclusions Our findings suggest that ARID1A protein transcriptionally modulates DUSP4 expression by remodeling chromatin, subsequently inactivating the MAPK pathway, leading to tumor suppression. The ARID1A-DUSP4-MAPK axis may be further considered for developing targeted therapies against ARID1A-mutated cancers.

Abstract Chemotherapy, radiotherapy, targeted therapy, and immunotherapy are established cancer treatment modalities that are widely used due to their demonstrated efficacy against tumors and favorable safety profiles or tolerability. Nevertheless, treatment resistance continues to be one of the most pressing unsolved conundrums in cancer treatment. Hypoxia-inducible factors (HIFs) are a family of transcription factors that regulate cellular responses to hypoxia by activating genes involved in various adaptations, including erythropoiesis, glucose metabolism, angiogenesis, cell proliferation, and apoptosis. Despite this critical function, overexpression of HIFs has been observed in numerous cancers, leading to resistance to therapy and disease progression. In recent years, much effort has been poured into developing innovative cancer treatments that target the HIF pathway. Combining HIF inhibitors with current cancer therapies to increase anti-tumor activity and diminish treatment resistance is one strategy for combating therapeutic resistance. This review focuses on how HIF inhibitors could be applied in conjunction with current cancer treatments, including those now being evaluated in clinical trials, to usher in a new era of cancer therapy.

Abstract Although endometriosis is primarily benign, it has been identified as a risk factor for endometriosis-associated ovarian cancer (EAOC). Genetic alterations in ARID1A, PTEN, and PIK3CA have been reported in EAOC; however, an appropriate EAOC animal model has yet to be established. Therefore, the present study aimed to create an EAOC mouse model by transplanting uterine pieces from donor mice, in which Arid1a and/or Pten was conditionally knocked out (KO) in Pax8-expressing endometrial cells by the administration of doxycycline (DOX), onto the ovarian surface or peritoneum of recipient mice. Two weeks after transplantation, gene KO was induced by DOX and endometriotic lesions were thereafter removed. The induction of only Arid1a KO did not cause any histological changes in the endometriotic cysts of recipients. In contrast, the induction of only Pten KO evoked a stratified architecture and nuclear atypia in the epithelial lining of all endometriotic cysts, histologically corresponding to atypical endometriosis. The induction of Arid1a; Pten double-KO evoked papillary and cribriform structures with nuclear atypia in the lining of 42 and 50% of peritoneal and ovarian endometriotic cysts, respectively, which were histologically similar to EAOC. These results indicate that this mouse model is useful for investigating the mechanisms underlying the development of EAOC and the related microenvironment.

Abstract Chromatin remodeling is an essential cellular process for organizing chromatin structure into either open or close configuration at specific chromatin locations by orchestrating and modifying histone complexes. This task is responsible for fundamental cell physiology including transcription, DNA replication, methylation, and damage repair. Aberrations in this activity have emerged as epigenomic mechanisms in cancer development that increase tumor clonal fitness and adaptability amidst various selection pressures. Inactivating mutations in AT-rich interaction domain 1A (ARID1A), a gene encoding a large nuclear protein member belonging to the SWI/SNF chromatin remodeling complex, result in its loss of expression. ARID1A is the most commonly mutated chromatin remodeler gene, exhibiting the highest mutation frequency in endometrium-related uterine and ovarian carcinomas. As a tumor suppressor gene, ARID1A is essential for regulating cell cycle, facilitating DNA damage repair, and controlling expression of genes that are essential for maintaining cellular differentiation and homeostasis in non-transformed cells. Thus, ARID1A deficiency due to somatic mutations propels tumor progression and dissemination. The recent success of PARP inhibitors in treating homologous recombination DNA repair-deficient tumors has engendered keen interest in developing synthetic lethality-based therapeutic strategies for ARID1A-mutated neoplasms. In this review, we summarize recent advances in understanding the biology of ARID1A in cancer development, with special emphasis on its roles in DNA damage repair. We also discuss strategies to harness synthetic lethal mechanisms for future therapeutics against ARID1A-mutated cancers.

ARID1A, which is often mutated in human endometrial cancer, is a component of the SWI/SNF chromatin remodelling complex. Here, the authors show that Arid1a mutations in the mouse endometrium and primary human endometrial epithelial cells cause widespread reprogramming of gene transcription and result in a loss of response to TGFβ.

Summary: PBX1 is a transcription factor involved in diverse cellular functions including organ development, stem cell renewal, and tumorigenesis. PBX1 is localized at chr1q23.3, a frequently amplified chromosomal region, and it is overexpressed in many human malignancies. Cancer cells with elevated PBX1 signaling are particularly vulnerable to PBX1 withdrawal. We designed a series of small molecule compounds capable of docking to the interface between PBX1 and its cognate DNA target sequence. Among them, T417 is found to be a lead compound. In cell-based assays, T417 significantly suppressed self-renewal and proliferation of cancer cells expressing high levels of PBX1. T417 also re-sensitized platinum-resistant ovarian tumors to carboplatin. T417 did not affect healthy tissues likely due to their lower PBX1 expression levels. Therefore, targeting PBX-DNA interface can be a promising strategy for treating human tumors reliant on PBX1 for survival.

Background: Spleen tyrosine kinase (SYK) is frequently upregulated in recurrent ovarian carcinomas, for which effective therapy is urgently needed. SYK phosphorylates several substrates, but their translational implications remain unclear. Here, we show that SYK interacts with EGFR and ERBB2, and directly enhances their phosphorylation. Methods: We used immunohistochemistry and immunoblotting to assess SYK and EGFR phosphorylation in ovarian serous carcinomas. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. To study its role in EGFR signaling, SYK activity was modulated using a small molecule inhibitor, a syngeneic knockout, and an active kinase inducible system. We applied RNA-seq and phosphoproteomic mass spectrometry to investigate the SYK-regulated EGF-induced transcriptome and downstream substrates. Findings: Induced expression of constitutively active SYK130E reduced cellular response to EGFR/ERBB2 inhibitor, lapatinib. Expression of EGFRWT, but not SYK non-phosphorylatable EGFR3F mutant, resulted in paclitaxel resistance, a phenotype characteristic to SYK active ovarian cancers. In tumor xenografts, SYK inhibitor reduces phosphorylation of EGFR substrates. Compared to SYKWT cells, SYKKO cells have an attenuated EGFR/ERBB2-transcriptional activity and responsiveness to EGF-induced transcription. In ovarian cancer tissues, pSYK (Y525/526) levels showed a positive correlation with pEGFR (Y1187). Intense immunoreactivity of pSYK (Y525/526) correlated with poor overall survival in ovarian cancer patients. Interpretation: These findings indicate that SYK activity positively modulates the EGFR pathway, providing a biological foundation for co-targeting SYK and EGFR. Fund: Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, NIH/NCI, Ovarian Cancer Research Foundation Alliance, HERA Women's Cancer Foundation and Roseman Foundation. Funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript and eventually in the decision to submit the manuscript.

Targeting histone acetylation modulators (HAMPs) is a promising avenue of drug discovery in cancer research. Here, the authors integrate multi-dimensional genomic profiles to systematically investigate recurrent genomic alterations in HAMPs, identifying potential therapeutic targets for precision epigenetic treatment.

Summary: Many gene products exhibit great structural heterogeneity because of an array of modifications. These modifications are not directly encoded in the genomic template but often affect the functionality of proteins. Protein glycosylation plays a vital role in proper protein functions. However, the analysis of glycoproteins has been challenging compared with other protein modifications, such as phosphorylation. Here, we perform an integrated proteomic and glycoproteomic analysis of 83 prospectively collected high-grade serous ovarian carcinoma (HGSC) and 23 non-tumor tissues. Integration of the expression data from global proteomics and glycoproteomics reveals tumor-specific glycosylation, uncovers different glycosylation associated with three tumor clusters, and identifies glycosylation enzymes that were correlated with the altered glycosylation. In addition to providing a valuable resource, these results provide insights into the potential roles of glycosylation in the pathogenesis of HGSC, with the possibility of distinguishing pathological outcomes of ovarian tumors from non-tumors, as well as classifying tumor clusters.

Summary: ARID1A, a subunit of the SWI/SNF complex, is among the most frequently mutated genes across cancer types. ARID1A is mutated in more than 50% of ovarian clear cell carcinomas (OCCCs), diseases that have no effective therapy. Here, we show that ARID1A mutation confers sensitivity to pan-HDAC inhibitors such as SAHA in ovarian cancers. This correlated with enhanced growth suppression induced by the inhibition of HDAC2 activity in ARID1A-mutated cells. HDAC2 interacts with EZH2 in an ARID1A status-dependent manner. HDAC2 functions as a co-repressor of EZH2 to suppress the expression of EZH2/ARID1A target tumor suppressor genes such as PIK3IP1 to inhibit proliferation and promote apoptosis. SAHA reduced the growth and ascites of the ARID1A-inactivated OCCCs in both orthotopic and genetic mouse models. This correlated with a significant improvement of survival of mice bearing ARID1A-mutated OCCCs. These findings provided preclinical rationales for repurposing FDA-approved pan-HDAC inhibitors for treating ARID1A-mutated cancers. : Fukumoto et al. show that ARID1A mutation confers sensitivity to pan-HDAC inhibitors such as SAHA in ovarian cancers. This correlated with enhanced growth suppression induced by the inhibition of HDAC2 activity in ARID1A-mutated cells. These findings provided preclinical rationales for repurposing FDA-approved pan-HDAC inhibitors for treating ARID1A-mutated cancers. Keywords: ovarian cancer, ARID1A, HDAC2, pan-HDAC inhibitor, SAHA, SWI/SNF, chromatin remodeling

It has previously been proposed that high-grade serous ovarian carcinoma (HGSOC) may originate from the fallopian tube. Here, the authors analyze genetic aberrances in fallopian tube lesions, ovarian cancers, and metastases from HGSOC patients and establish the evolutionary origins of HGSOC in the fallopian tube.

High-grade serous carcinomas (HGSCs) are associated with precursor lesions (STICs) in the fallopian epithelium in only half of the cases. Here the authors report the molecular analysis of HGSCs with and without associated STICs and show similar profiles supporting a common origin for all HGSCs.

The CINdex Bioconductor package addresses an important area of high-throughput genomic analysis. It calculates the chromosome instability (CIN) index, a novel measurement that quantitatively characterizes genome-wide copy number alterations (CNAs) as a measure of CIN. The advantage of this package is an ability to compare CIN index values between several groups for patients (case and control groups), which is a typical use case in translational research. The differentially changed cytobands or chromosomes can then be linked to genes located in the affected genomic regions, as well as pathways. This enables in-depth systems biology–based network analysis and assessment of the impact of CNA on various biological processes or clinical outcomes. This package was successfully applied to analysis of DNA copy number data in colorectal cancer as a part of multi-omics integrative study as well as for analysis of several other cancer types. The source code, along with an end-to-end tutorial, and example data are freely available in Bioconductor at http://bioconductor.org/packages/CINdex/ .

Manipulating gene expression in mammalian cell lines is one of the most widely used methods for studying gene function. Tetracycline- and doxycycline-inducible systems are sensitive, reproducible, relatively inexpensive, and proven to work well in both cell lines and mouse models. However, obtaining homogeneous transgene expression or uniform knockdown by short hairpin RNA requires time-consuming and labor-intensive single-cell cloning to derive stable cell lines. For this reason, Tet-inducible cell systems have yet to be widely adopted. Here we describe the XT-cell method, a novel system for establishing isogenic inducible cell lines using founder reporter lines and recombinase-mediated cassette exchange. We demonstrate that, using this XT-cell method, isogenic stable Tet-inducible cell lines can be efficiently created with much less effort and time as compared with conventional methods. The XT-plasmids and the XT-founder cell lines will be a valuable resource to researchers interested in versatile modulation of gene expression in cell culture systems, and this method has the potential to expedite many aspects of biomedical research.

We determined whether the mutations found in ovarian cancers could be identified in the patients' ovarian cyst fluids. Tumor-specific mutations were detectable in the cyst fluids of 19 of 23 (83%) borderline tumors, 10 of 13 (77%) type I cancers, and 18 of 18 (100%) type II cancers. In contrast, no mutations were found in the cyst fluids of 18 patients with benign tumors or non-neoplastic cysts. Though large, prospective studies are needed to demonstrate the safety and clinical utility of this approach, our results suggest that the genetic evaluation of cyst fluids might be able to inform the management of the large number of women with these lesions.

There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 - 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 - 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations.

[This corrects the article DOI: 10.1371/journal.pone.0156069.].

The role of the forkhead box Q1 (FOXQ1) transcription factor in cancer pathogenesis has recently emerged. Overexpression of FOXQ1 has been found in a variety of human cancers, and its upregulation has been associated with poor prognosis in colorectal, breast, and non-small cell lung carcinomas. However, the molecular mechanism underlying how FOXQ1 contributes to ovarian epithelial carcinomas remains unclear. To this end, we analyzed gene expression levels in ovarian cancer tissues and cell lines and demonstrated a higher expression level of FOXQ1 in epithelial ovarian cancer cells than that in normal epithelial cells. We then used a human ovarian cancer cell line, SKOV3, which expressed a higher level of FOXQ1, as a cell model to investigate the biological effects of FOXQ1 by using RNA interference. Silencing of FOXQ1 expression using a shRNA knockdown approach affected the expression of several cell cycle regulators, leading to suppressed cell proliferation, reduced cell motility/invasion, and upregulation of epithelial cell markers and the downregulation of mesenchymal cell markers. Taken together, these results suggest that FOXQ1 expression is essential to maintain cell proliferation, motility/invasion, and epithelial-mesenchymal transition phenotypes in ovarian cancer cells.

AT-rich interactive domain 1A (ARID1A) has emerged as a new tumor suppressor in which frequent somatic mutations have been identified in several types of human cancers. Although most ARID1A somatic mutations are frame-shift or nonsense mutations that contribute to mRNA decay and loss of protein expression, 5% of ARID1A mutations are in-frame insertions or deletions (indels) that involve only a small stretch of peptides. Naturally occurring in-frame indel mutations provide unique and useful models to explore the biology and regulatory role of ARID1A. In this study, we analyzed indel mutations identified in gynecological cancers to determine how these mutations affect the tumor suppressor function of ARID1A. Our results demonstrate that all in-frame mutants analyzed lost their ability to inhibit cellular proliferation or activate transcription of CDKN1A, which encodes p21, a downstream effector of ARID1A. We also showed that ARID1A is a nucleocytoplasmic protein whose stability depends on its subcellular localization. Nuclear ARID1A is less stable than cytoplasmic ARID1A because ARID1A is rapidly degraded by the ubiquitin-proteasome system in the nucleus. In-frame deletions affecting the consensus nuclear export signal reduce steady-state protein levels of ARID1A. This defect in nuclear exportation leads to nuclear retention and subsequent degradation. Our findings delineate a mechanism underlying the regulation of ARID1A subcellular distribution and protein stability and suggest that targeting the nuclear ubiquitin-proteasome system can increase the amount of the ARID1A protein in the nucleus and restore its tumor suppressor functions.

Recent genome-wide analysis has demonstrated that somatic mutations in ARID1A (BAF250) are the most common molecular genetic changes in ovarian clear cell carcinoma (OCCC). ARID1A mutations, which occur in approximately half of OCCC cases, lead to deletion of the encoded protein and inactivation of the putative tumor suppressor. In this study, we determined the significance of loss of ARID1A immunoreactivity with respect to several clinicopathological features in a total of 149 OCCCs. First, we demonstrated that ARID1A immunohistochemistry showed concordance with the mutational status in 91% of cases with 100% sensitivity and 66% specificity. Specifically, among 12 OCCC cases for which ARIDA mutational status was known, ARIDIA immunoreactivity was undetectable in all 9 cases harboring ARID1A mutations and was undetectable in one of 3 cases with wild-type ARID1A. With respect to the entire cohort, ARID1A immunoreactivity was undetectable in 88 (59%) of 149 OCCCs. There was no significant difference between ARID1A negative and positive cases in terms of histopathologic features, age, clinical stage, or overall survival. In conclusion, this study provides further evidence that mutations in ARID1A resulted in loss of ARID1A protein expression in OCCC, although no significant differences between ARID1A positive and negative cases were observed with respect to any clinicopathological features examined.

Acquisition of novel gene products or new antigens in cancer cells elicits a host immune response that results in selection pressure for tumor clones to evade immunosurveillance. Similar to maternal–fetal tolerance and allotransplantation acceptance, upregulation of HLA-G expression has been found as one of the mechanisms that are programmed in cancer cells. HLA-G expression is frequently detected in a wide variety of human cancers and its protein levels negatively correlate with poor clinical outcome. The immune inhibitory effect can be achieved by binding of HLA-G molecules to the immunoglobulin-like inhibitory receptors that are expressed on the immunocompetent cells at all stages of the immune response. This review summarizes recent studies of HLA-G expression in human cancer, with a special focus on the molecular mechanisms that underlie how HLA-G molecules facilitate tumor cell evasion of the host immune response, and presents new directions for developing HLA-G-based diagnosis/therapeutics.

Ovarian cancer patients treated with cisplatin-based chemotherapy often develop acquired cisplatin resistance and, consequently, cancer recurrence. We have previously reported that annexin A11 is associated with cisplatin resistance and related to tumor recurrence in ovarian cancer patients. In this study, we used small interfering RNA to suppress annexin A11 expression in ovarian cancer cells followed by various in vitro assays. We showed that knockdown of annexin A11 expression reduced cell proliferation and colony formation ability of ovarian cancer cells. Epigenetic silencing of annexin A11 conferred cisplatin resistance to ovarian cancer cells. Through a comprehensive time course study of cisplatin response in ovarian cancer cells with/without suppression of annexin A11 expression using whole-genome oligonucleotide microarrays, we identified a set of differentially expressed genes associated with annexin A11 expression and some patterns of gene expressions in response to cisplatin exposure. These identified genes/patterns were further validated by real-time polymerase chain reaction and immunoblot analysis. Many of them such as HMOX1, TGFBI, LY6D, S100P, EIF4EBP2, DHRS2, and PCSK9 have been involved in apoptosis, cell cycling/proliferation, cell adhesion/migration, transcription regulation, and signal transduction. In addition, immunohistochemistry analyses indicated that annexin A11 immunointensity inversely correlated with HMOX1 immunoreactivity in 142 ovarian cancer patients. In contrast to annexin A11, HMOX1 immunoreactivity positively correlated with in vitro cisplatin resistance in ovarian cancers. Collectively, annexin A11 is directly involved in cell proliferation and cisplatin resistance of ovarian cancer. Manipulation of annexin A11 and its associated genes may represent a novel therapeutic strategy in human ovarian cancers.

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

NAC1 encoded by NACC1 is a member of the BTB/POZ family of proteins and participates in several pathobiological processes. However, its function during tissue development has not been elucidated. In this study, we compared homozygous null mutant Nacc1(-/-) and wild type Nacc1(+/+) mice to determine the consequences of diminished NAC1 expression. The most remarkable change in Nacc1(-/-) mice was a vertebral patterning defect in which most knockout animals exhibited a morphological transformation of the sixth lumbar vertebra (L6) into a sacral identity; thus, the total number of pre-sacral vertebrae was decreased by one (to 25) in Nacc1(-/-) mice. Heterozygous Nacc1(+/-) mice had an increased tendency to adopt an intermediate phenotype in which L6 underwent partial sacralization. Nacc1(-/-) mice also exhibited non-closure of the dorsal aspects of thoracic vertebrae T10-T12. Chondrocytes from Nacc1(+/+) mice expressed abundant NAC1 while Nacc1(-/-) chondrocytes had undetectable levels. Loss of NAC1 in Nacc1(-/-) mice was associated with significantly reduced chondrocyte migratory potential as well as decreased expression of matrilin-3 and matrilin-4, two cartilage-associated extracellular matrix proteins with roles in the development and homeostasis of cartilage and bone. These data suggest that NAC1 participates in the motility and differentiation of developing chondrocytes and cartilaginous tissues, and its expression is necessary to maintain normal axial patterning of murine skeleton.

PBX1 is a TALE homeodomain transcription factor involved in organogenesis and tumorigenesis. Although it has been shown that ovarian, breast, and melanoma cancer cells depend on PBX1 for cell growth and survival, the molecular mechanism of how PBX1 promotes tumorigenesis remains unclear. Here, we applied an integrated approach by overlapping PBX1 ChIP-chip targets with the PBX1-regulated transcriptome in ovarian cancer cells to identify genes whose transcription was directly regulated by PBX1. We further determined if PBX1 target genes identified in ovarian cancer cells were co-overexpressed with PBX1 in carcinoma tissues. By analyzing TCGA gene expression microarray datasets from ovarian serous carcinomas, we found co-upregulation of PBX1 and a significant number of its direct target genes. Among the PBX1 target genes, a homeodomain protein MEOX1 whose DNA binding motif was enriched in PBX1-immunoprecipicated DNA sequences was selected for functional analysis. We demonstrated that MEOX1 protein interacts with PBX1 protein and inhibition of MEOX1 yields a similar growth inhibitory phenotype as PBX1 suppression. Furthermore, ectopically expressed MEOX1 functionally rescued the PBX1-withdrawn effect, suggesting MEOX1 mediates the cellular growth signal of PBX1. These results demonstrate that MEOX1 is a critical target gene and cofactor of PBX1 in ovarian cancers.

Ovarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients.We compared the proteomes of paired primary and recurrent post-chemotherapy ovarian high-grade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-kappaB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-kappaB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in naïve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells.Proteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-kappaB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of cancers, and these genes are thought to function as both tumor suppressors and oncogenes.Using microRNA microarrays, we identify several miRNAs aberrantly expressed in human ovarian cancer tissues and cell lines. miR-221 stands out as a highly elevated miRNA in ovarian cancer, while miR-21 and several members of the let-7 family are found downregulated. Public databases were used to reveal potential targets for the highly differentially expressed miRNAs. In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels. Interestingly, there was little overlap between the predicted and the experimental targets or pathways, or between experimental targets/pathways obtained using different cell lines, highlighting the complexity of miRNA target selection.Our results identify several differentially expressed miRNAs in ovarian cancer and identify potential target transcripts that may be regulated by these miRNAs. These miRNAs and their targets may have important roles in the initiation and development of ovarian cancer.